Over the past 100 years, the development and widespread use of vaccines against infectious agents has been one of the triumphs of medical science. One reason for the success of these vaccines is that they excel at inducing antibody production (humoral immunity), which are the principle components of immune protection against most viruses and bacteria. There are, however exceptions, including medically important intracellular organisms like Mycobacterium tuberculosis, the malaria parasite, Leishmania parasite, and possibly the human immunodeficiency virus (HIV), in which protection depends more on cell-mediated immunity than on the induction of antibodies (humoral immunity).
The conventional active vaccines are made either of a killed or attenuated form of the infectious agent. Also a modified product of the infectious agent (toxoid) or a constituent of an infectious agent (such as the capsule) is used. These vaccines have some limitations and problem with the antibody response they induce. Moreover, large and repeated doses are required to administer when a non-viable (killed, attenuated organism, toxoid or capsule) vaccine is used and the protective immunity obtained is not long lasting. Furthermore, the process of manufacturing live attenuated vaccine and killed vaccines can alter the structure of native protein and thus lower the antigenecity of the vaccine and in most cases usually a humoral but not a cell-mediated immune response is generated. What is required in such cases, but not available, are antigens that are safe to use, that can be processed by the endogenous pathway and eventually activate cytotoxic T-lymphocytes (CTL). This becomes highly desirable for intracellular pathogens such as Leishmania parasite. The activated CTL generated in this way would destroy the parasite-infected cell.
For these reasons, new approaches of vaccination are under intensive investigations that involve the injection of a piece of DNA that contains the gene for the antigen of interest. Various recent reports of induction of cellular immune responses by a DNA vaccine against various parasites including Plasmodium and Leishmania, and to various bacterial species such as Mycobacterium spp in humans raises hope for the clinical applicability of this method of immunization.
In a DNA vaccine, the gene for the desired antigen (pathogen origin) of interest is cloned into a bacterial plasmid that is engineered to augment the expression of the inserted gene in mammalian cells. After being injected into an animal, the plasmid enters host cells, where it remains in the nucleus as an episome; without getting integrated into the host cell DNA. Using the host cell's metabolic machinery, the inserted clone DNA in the episome directs the synthesis of the antigen it encodes.
An approach involving the synthesis of antigen within the cells has several potential advantages over immunization with exogeneous recombinant proteins or killed organisms. A protein produced by plasmid-transfected cells is likely to be folded in its native configuration, which favors the production of neutralizing antibodies. Furthermore, the peptide synthesized under the direction of the plasmid DNA can be brought to the surface of cells and displayed by MHC class I molecules, an essential step in the stimulation of CD8+ cytotoxic T cells, which further evokes cell mediated immunity. On contrast, standard vaccine antigens are taken up into cells by phagocytosis or endocytosis and are processed through the MHC class II system, which primarily stimulates antibody response. Finally, DNA vaccines have be shown to persist and stimulate sustained immune responses.
In addition to being able to induce the appropriate immune responses, DNA vaccines are also attractive because they ensure appropriate folding of the polypeptide, produce and release the antigen over long periods, and do not require adjuvants. Other advantages include the stability of the DNA molecule, long shelf life, and do not require a strict cold chain for distribution. DNA vaccines are also safer than certain live-virus vaccines, for use in immuno compromised patients such as those infected with HIV. It also bypasses the numerous problems associated with conventional vaccines, such as immune responses against the delivery vector and concerns about safety related to the use of viral vectors. They DNA vaccines can be constructed in such a way that the genes from different pathogens are included in the same plasmid, thus potentially decreasing the number of vaccinations required for children. Moreover in tropical countries like ours where people usually suffer from more than one type of infection, chimeric vaccines have become an important need.
Both Leishmania and Mycobacterium are considered to be important human pathogen. The world health organization (WHO) considers leishmaniasis to b one of the important parasitic diseases with approximately 350 million people at risk of contracting the disease. The disease has worldwide distribution, and is endemic in at least 88 countries, and the disease occurs on all continents except Antarctica were no suitable vectors are present. On the other hand Mycobacterial infections remain major cause of mortality and morbidity worldwide. Tuberculosis causes 2-3 millions deaths and 15 million new cases per annum worldwide, while more than 1000 people die off every day in India.
Considering the treatment, drug resistant tuberculosis and drug resistant leishmaniasis have become a major health problem. Particularly of interest is multidrug resistance tuberculosis, where the patients become insensitive to different drugs. Today treatment of leishmaniasis also constitutes a difficult challenge due to co-infection with HIV and resistance to pentavalent antimonials (most common, affordable drug against leishmaniasis). The problem becomes more severe due to poverty and malnutrition, migration of non-immune refugees, insufficient diagnostic tools and unavoidable or unaffordable drugs.
While the relationship between HIV/TB and HIV/Leishmaniasis has been documented, little is known about TB/Leishmania co-infection, a syndrome that has important clinical implications. Although distinct in aetiology and transmission mechanisms, VL and TB share several features. The most important is that both are intracellular in nature and cell mediated immunity plays important role in protection against infection. Moreover, in both cases infectious remain asymptomatic in several infected persons. Symptoms usually develop after several months or years and progress to clinical disease. Very long incubation periods (latent infection) may be related to immune suppression occurring at a later age, which apparently turns the latent infection into active disease.
It is reported that Tuberculosis (TB) can cause immunosuppression by blocking macrophage response to IFN-γ by inhibiting the transcription of IFN-γ-responsive genes, which results in the progression of latent leishmanial infection to clinical manifestation. Mycobacterium also involves in the down-regulation of the Ag-presenting molecule CD1 from the cell surface of CD1+ APCs. The loss of CD1 from the cell surface is associated with a complete inhibition of the ability of the infected cells to present Ag to CD1-restricted dendritic cells, which can initiate antimicrobial responses by CD1-mediated presentation of pathogen-derived glycolipids.
Similarly, VL can reactivate a latent mycobacterial infection, Leishmania is known to downmodulate Nitric Oxide production, correlated with a reduction in inducible nitric oxide synthase (iNOS) activity. Leishmania like Mycobacteria inhibits CD1 expression and prevents activation of CD1-restricted T cells by dendritic cells. Evasion of presentation by CD1 may represent a Leishmania survival strategy to avoid recognition of abundant parasite glycolipids. Moreover, it have also been observed that Leishmania infected macrophages are less efficient at promoting the sustained TCR signaling necessary for activation of T cell and for IFN-γ production.
In the present situation it has become imperative to control co-infection cases particularly in areas where both leishmaniasis and tuberculosis occur concomitantly. The development of chimeric vaccine will provide an effective strategy in controlling leishmaniasis and Mycobacterial infection and further reducing the number of vaccination required. Moreover, vaccinations have an added advantage of avoiding the problem encountered with drug resistance.
As far as development of various specific vaccines is concerned for leishmaniasis numerous antigens have been tested with variable success rates using in vitro and mouse models. Among the various vaccines developed to date, heat killed vaccines are the most popular one. The first study was of a double blind randomized trial in which a pool of five strains of merthiolate-killed L. major was used as vaccine. The results showed that the protection against cutaneous Leishmaniasis was 23% and 60% in placebo and vaccine arms, respectively. Similar strategy was used in India also at CDRI Lucknow, using heat killed (autoclaved) L. donovani along with Mycobacterium habana and L. major along with BCG were used as a vaccine against visceral and cutaneous leishmaniasis. The vaccine shows good protective efficacy but the major problem with the vaccine is that the antigen alone produces only marginal protection and requires BCG as adjuvant; moreover it causes localized allergic reaction at site of inoculation. Although the single dose of vaccine was protective in monkeys. However, all animals did have occasional parasites, even on day 90 post inoculation.
In Iran, a mixed BCG L. major killed vaccine has also undergone clinical trials for safety and efficacy. Although it proved safe, its efficacy was only 35%.
The relative merits of live-attenuated vaccines versus killed vaccines have been a constant subject of debate in relation to many antimicrobial and viral vaccines. The most notable arguments have been those concerned with immunogenicity, efficacy, safety, ease of production and distribution, and stability. The absence of a clear genetic profile of any Leishmania strain that can be labeled as a virulent, has kept the possibility of using live attenuated vaccine at abeyance. But recent advances to manipulate the Leishmania genome by introducing or eliminating genes have opened the avenues and potential to generate live-attenuated vaccines. It is now possible to generate parasites lacking genes essential for long-term survival in the mammalian host, e.g. by deleting the gene encoding the enzyme dihydrofolate reductase-thymidylate synthetase (DHFR-TS). In a mouse model, L. major parasites lacking DHFR-TS induce protection against the infection of L. major and L. amazonensis. However the disadvantages of such vaccine are its large-scale production and distribution in the field. Also no study has been done to evaluate its protection efficacy against L. donovani. 
The newer vaccines under consideration comprise of recombinant DNA-derived antigens and peptides. Some of the target antigens are species ad life cycle stage specific, while others are shared by promastigotes and amastigotes. Some are conserved among Leshmania species, while others are not. Also another biggest advantage is that these vaccines can be delivered as purified immunogens, because the naked DNA encodes them. Genetic manipulations can also allow us to target these antigens to specific locations or to particular antigen-presenting cells, such as dendritic cells or Langerhans cells, which are considered to be essential for the initiation of primary T-cell responses.
The first recombinant antigen used as vaccine against leishmaniasis was leishmaniolysin or gp63. This is a membrane ecto-metalloprotease present in promastigotes of all species. The gp63 is also one of the parasite receptors for host macrophages, and parasite mutants lacking the protein are reported to be avirulent. Gp63 is known to help promastigotes by rendering them resistant to complement-mediated cytolysis. It also appears to act, (perhaps, together with LPG), namely infection of macrophages by promastigotes via receptor-mediated endocytosis. However, the efficacy was found to be only 50% and that too at higher concentration. Several factors may account for this. The antigen needs to be in their native conformation for processing, and Escherichia coli-derived recombinant proteins may not fulfill this requirement. Another reason for the low success rate could be that some polypeptides might be minor immunogen only. Apart from gp63, other antigens like LACK and A2 were also tested, but the results are not satisfactory. Failure of this single antigen vaccines leads to development of conjugate polypeptide vaccines. One such candidate is recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with monophosphoryl lipid A-squalene (MPL-SE) suitable for human use. This vaccine candidate is the first defined vaccine for cutaneous leishmaniasis in human clinical trials and has completed phase 1 and 2 safety and immunogenecity testing in normal, healthy human subjects. The vaccine candidate also show good protective efficacy against visceral leishmaniasis in hamster model.
Naked DNA vaccines have become popular recently and revolutionized the prevention and treatment strategies against infectious diseases, particularly against viruses and bacteria. However, DNA vaccines have not made much in-road in the field of medical parasitology except against malaria, to some extent. In India work on DNA vaccine against Leishmania donovani is in progress. Vaccination with ORFF gene induced both humoral and cellular immune response against ORFF, which provided a significant level of protection against challenge with L. donovani in mouse model. However no further studies involving efficacy studies in primates model or phase trials have been done for its application in human.
For developing vaccine against Mycobacterium, immunization work is done mainly on the vaccine strain of M. bovis [Bacilli Calmette-Guerin (BCG)] that provides partial but variable, protection against tuberculosis and leprosy. BCG is derived from attenuation of an isolated strain of Mycobacterium bovis. It was introduced as a tuberculosis vaccine for humans in 1921 and has been relatively safe with rare incidences of adverse reactions. BCG vaccination has been shown in some studies to effectively boost the immune response against primary infection but has limited effect on subsequent course of dormancy and reactivation. Little or no protection is seen after 10 to 15 years which suggests that childhood vaccination will not prevent adult re-infection.
Safety of the BCG vaccine is a growing concern after epidemic of HIV. In order to avoid potential adverse effects of BCG within immunocompromised individuals, BCG auxotrophs have been developed using the previously mentioned technique. Auxotrophs are mutants that require a specific nutrient or metabolite that is not required by the wild type. As a result, such mutants can only survive for a short period of time within a host, if the host lacks the specified nutrient. Five such strains were tested in mice with severe combined immunodeficiency disease (SCID) for safety, and in a susceptible strain of mice for protection. Results have shown that these strains are safe in SCID mice, and demonstrate the same amount of protective immunity as normal BCG in susceptible mice, suggesting that this could be a safer method of vaccination.
An innovative vaccine approach currently being applied in the search for a BCG replacement is the protein or DNA vaccine. This includes a number of protein and DNA molecules expressing single Mycobacterial antigen that could induce partial protection against experimental infection with M. tuberculosisl. Some of these antigens are those proteins that are secreted by Mycobacterium during their residence in macrophages, such as: i) the antigen 85 complex of proteins (85A, 85B, 85C), ii) a 6 kDa protein termed Early Secreted Antigenic Target (ESAT-6) , iii) a 38 kDa lipoprotein with homology to PhoS, iv) the 65 kDa heat-shock protein (Hsp 65) v) a 55 kDa protein rich in proline and threonine and vi) a 19 kDa lipoprotein.
The applicant also did an extensive search and found that although various vaccine candidates have been developed against visceral leishmaniasis and tuberculosis but none of them was developed as chimera of L. donovani and M tuberculosis with SEQ ID No. 1 and SEQ ID No. 2.
Discussed below are the few US patents on chimeric constructs, DNA and recombinant polypeptide vaccine constructs on the subject concerned and the uniqueness of the applicants' construct.
The disclosed a chimera of polypeptide from Leishmania infantuml. However the present invention is chimera of polynucleotide.
The WIPO Patent no. WO/2006/053485 by Zhongming, in Nov. 14, 2005 teaches about a chimeric mycobacterium tuberculosis gene vaccine and the preparation method thereof. A chimeric mycobacterium tuberculosis gene vaccine is provided. The vaccine comprises Ag85a gene encoding a structural protein of Mycobacterium tuberculosis and ESAT6 gene of Mycobacterium tuberculosis, wherein the ESAT6 gene is inserted into the sequence of Ag85a gene, and the Ag85a gene is inserted to eukaryotic expression vector pVAX1. The composition of the invention is useful for inducing the immune system in mice and monkey.
The above disclosed a chimera vaccine for control of tuberculosis only. Moreover, the antigen the chimeric construct express was a conjugated chimeric peptide. However, the present invention provides chimera of Kinesin motor domain and esat-6 gene useful in control of leishmaniasis and tuberculosis both. Also the construct is designed in such a way that it allows individual expression of both the gene from chimeric construct, instead of generation of chimeric peptide.
The European Patent No. EP1279679 by Soto at al., in Jan. 29, 2003 teaches about A composition and method for stimulating an immune response against an antigen in immunized individuals or in cell groups. The composition comprises a protein called Lip2a Leishmania formed by a sequence of amino acids coded for by a sequence of DNA. The compositions of the invention are useful for promoting a humoral or cellular response in the individual who is inoculated with said compositions. The invention does not deal with either chimera molecular or with SEQ ID No. 1 and SEQ ID No. 2
The U.S. Pat. No. 5,674,503 by Olafson in Oct. 7, 1997 teaches about Peptides capable of eliciting an immune response to leishmaniasis and methods of using the same. The invention provides a pharmaceutical composition comprising the peptides or immunogens, in combination with a physiologically acceptable carrier or diluent. However applicant's present invention does no deal with peptides.
The U.S. Pat. No. 5,736,524 by Content et al., in Apr. 7, 1998 teaches about Polynucleotide tuberculosis vaccine. This invention provides DNA construct which, when directly introduced into a vertebrate in vivo, including mammals such as humans induces the expression of encoded proteins within the animal. The result, as shown in this disclosure, is induction of immune responses against M.tb. Polynucleotides for the purpose of generating protective immune responses against M.tb infection.
The above although disclosed a DNA vaccine but it does deal with chimera. Moreover it is useful for control of tuberculosis only.